Purified culture of streptomyces galanosa

ABSTRACT

Acids, esters, imine acids, imine esters, and imine amides derived from 6-formyl-4,7,9-trihydroxy-1-phenazinecarboxylic acid, methyl ester and from 6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methyl ester demonstrate anti-microbial and antineoplastic activity. 
     A purified strain of Streptomyces galanosa, NRRL 15738, is capable of producing 6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methyl ester in isolable quantities from a fermentation broth containing assimilable sources of carbon and nitrogen.

This application is a divisional of U.S. Ser. No. 743,321 filed June 10,1985, now U.S. Pat. No. 4,657,909.

BACKGROUND OF THE INVENTION

The present invention relates to novel phenazine carboxaldehydes andderivatives having antimicrobial and antineoplastic activity, to methodsof preparing the novel compounds, to a purified strain of Streptomycesgalanosa NRRL 15738 useful in the preparation of starting material forcompounds of the present invention, and to a method for the use ofcompounds of the present invention as antimicrobial or antineoplasticagents.

The present invention also relates to novel compounds useful asintermediates in the preparation of the phenazine carboxaldehydederivatives of the invention.

Phenazine compounds are known as natural products (see J. M. Ingram, etal, Adv. Appl. Microbiol., 13:267 (1970)), includingphenazine-1-carboxamide (oxychloraphine), and methoxylated derivativesof phenazine-1-carboxylic acid (e.g., griseolutin).

Phenazine-1-carboxylic acid has been evaluated as an antitumor agent(see V. Chernetskii et al, Onkologyia (Kiev), 5:110 (1974) and O. A.Sidorik et al, Fiziol. Act. Veshch, 6:92 (1974)).

Substituted phenazine-1-carboxamides have also been evaluated forantitumor activity, and found active against sarcoma 180 (see K.Katagiri et al, Kenyu Nempo, 17:127 (1967)).

Phenazine-1-carboxamides bearing a tetrazole side chain have beenevaluated as antiallergy agents (U.S. Pat. No. 4,337,579).

3,4-Benzophenazine-1-carboxamides bearing a variety of side chainsincluding dimethylaminoethyl have been evaluated for antimicrobialactivity (see H. Shoji et al, Yakugaku Zasshi, 103:245 1982)).

Phenazine-1,4-dicarboxamides bearing alkylamino-and dialkylaminopropylside chains have been evaluated for antimalarial activity (see S. N.Sawhney et al, J. Pharm. Sci., 68:524 (1979)).

The natural phenazine derivative, lomondomycin (also known in theliterature as lomofungin), is reported as being produced in fermentationbroths by the microorganism Streptomyces lomondensis NRRL 3252 (U.S.Pat. No. 3,359,165) and has been assigned the structure corresponding to6-formyl-4,7,9-trihydroxy-1-phenazinecarboxylic acid, methyl ester (seeC. G. Tipton, et al, J. Amer. Chem. Soc., 92(5):1425-1426 (1970)).

SUMMARY OF THE INVENTION

In its broadest aspect, the present invention provides compounds havingthe structural Formula I ##STR1## wherein R₁ is hydrogen or methyl; R₂is oxygen, or ═N--Y--NR₄ R₅, where Y is cyclohexyl or a straight orbranched alkylene group of from one to six carbons optionallysubstituted with hydroxyl; R₄ and R₅ are independently hydrogen, alkylof from one to four carbon atoms, optionally substituted with hydroxyl;or alternatively, R₂ is ═N(CH₂)_(n) --W where n equals zero to four andW is a pyrrolidinyl or piperidinyl ring optionally substituted withalkyl of from one to four carbon atoms which may be further substitutedwith hydroxyl; R₃ is hydroxy, alkoxy of from one to four carbon atoms,or --NH--Y'--NR₄ 'R₅ ' where Y' is cyclohexyl or a straight or branchedalkylene group of from one to to six carbon atoms optionally substitutedwith hydroxyl; and R₄ ' and R₅ ' are independently hydrogen, alkyl offrom one to four carbon atoms, optionally substituted with hydroxyl; oralternatively, R₃ is --NH(CH₂)_(n) '--W' where n' equals zero to fourand W' is a pyrrolidinyl or piperidinyl ring optionally substituted withalkyl of from one to four carbon atoms which may be further substitutedwith hydroxyl; with the provisos that (a) when R₂ is ═N--Y--NR₄ R₅ andR₃ is --NH--Y'--R₄ 'R₅ ' the groups Y and Y' may be the same ordifferent, R₄ and R₄ ' may be the same or different, and R₅ and R₅ ' maybe the same or different, or, alternatively, when R₂ is ═N--(CH₂)_(n)--W and R₃ is --NH--(CH₂)_(n) '--W' the numbers n and n' may be the sameor different and the groups W and W' may be the same or different; and(b) when R₁ is hydrogen and R₂ is oxygen, R₃ may not be hydroxy oralkoxy; and the pharmaceutically acceptable salts thereof.

In another aspect of the present invention there are providedpharmaceutical compositions useful for the treatment of microbialinfections in a mammal comprising an antimicrobially effective amount ofa compound of Formula I in combination with a pharmaceuticallyacceptable carrier.

In yet another aspect, the present invention provides a method oftreating microbial infections in a mammal comprising administering to amammal in need of such treatment a pharmaceutical composition comprisingan antimicrobially effective amount of a compound of Formula I incombination with a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a method of inhibitingthe growth of neoplasms in a mammal comprising the administration to amammal in need of such treatment a pharmaceutical composition comprisingan antineoplastically effective amount of a compound of Formula I incombination with a pharmaceutically acceptable carrier.

In yet another aspect, the present invention provides a pure strain ofStreptomyces galanosa, designated NRRL 15738, which is capable ofproducing 6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid,methyl ester in isolable quantities from a fermentation broth containingassimilable sources of carbon and nitrogen under conditions of aerobicfermentation.

In a further aspect, the present invention provides a method ofpreparing a compound having the structural formula ##STR2## wherein R₁is hydrogen or methyl; R₂ is oxygen, or ═N--Y--NR₄ R₅, where Y iscyclohexyl or a straight or branched alkylene group of from one to sixcarbons optionally substituted with hydroxyl; R₄ and R₅ areindependently hydrogen, alkyl of from one to four carbon atoms,optionally substituted with hydroxy;, or alternatively, R₂ is═N(CH₂)_(n) --W where n equals zero to four and W is a pyrrolidinyl orpiperidinyl ring optionally substituted with alkyl of from one to fourcarbon atoms which may be further substituted with hydroxyl; R₃ ishydroxy, alkoxy of from one to four carbon atoms or atoms, or--NH--Y'--NR₄ 'R₅ ' where Y' is cyclohexyl or a straight or branchedalkylene group of from one to six carbon atoms optionally substitutedwith hydroxyl; and R₄ ' and R₅ ' are independently hydrogen, alkyl offrom one to four carbon atoms, optionally substituted with hydroxyl; or,alternatively, R₃ is --NH(CH₂)_(n) '--W' where n' equals zero to fourand W' is a pyrrolidinyl or piperidinyl ring optionally substituted withalkyl of from one to four carbon atoms which may be further substitutedwith hydroxyl; with the provisos that (a) when R₂ is ═N--Y--NR₄ R₅ andR₃ is --NH--Y'--R₄ 'R₅ ' the groups Y and Y' may be the same ordifferent, R₄ and R₄ ' may be the same or different, and R₅ and R₅ ' maybe the same or different; or, when R₂ is ═N(CH₂)_(n) --W and R₃ is--NH(CH₂)_(n) '--W', the numbers n and n' may be the same or different,and the groups W and W' may be the same or different; and (b) when R₁ ishydrogen and R.sub. 2 is oxygen, R₃ may not be hydroxy or alkoxy; andthe pharmaceutically acceptable salts thereof, comprising the steps of

(a) reacting a compound having the structural Formula II ##STR3##wherein R₁ is hydrogen or methyl and R₃ is hydroxy or alkoxy with oneequivalent of a compound having the formula R₄ R₅ N--Y--NH₂ orW--(CH₂)_(n) --NH₂ where R₄, R₅, Y, n, and W are as previously definedto form an imine ester or imine carboxylic acid compound having thestructural Formula IIIa or IIIb ##STR4## where R₃ is hydroxy or alkoxyand Y, R₄, R₅, n, and W are as previously defined, or

(b) alternatively converting a compound of structural Formula II whereR₃ is methoxy to a higher alkyl ester where R₃ is alkoxy of from two tofour carbon atoms by conventional transesterification methods andthereafter converting, if desired, the resulting higher alkyl ester toan imine ester of Formula IIIA or IIIB by the process of step (a);

(c) alternatively thereafter saponifying the imine ester product of step(a) or step (b), if desired, with aqueous base to form the correspondingimine carboxylic acid compound; or alternatively

(d) reacting the imine ester product of step (a) or step (b) with acompound having the formula R₄ 'R₅ 'N--Y'--NH₂ where Y' is cyclohexyl ora straight or branched alkylene group of from one to six carbon atomsoptionally substituted with hydroxyl; and R₄ ' and R₅ ' areindependently hydrogen, alkyl of from one to four carbon atoms,optionally substituted with hydroxyl, or with a compound having theformula W'--(CH₂)_(n) '--NH₂ where n' equals zero to four and W' is apyrrolidinyl or piperidinyl ring optionally substituted with alkyl offrom one to four carbons atoms which may be further substituted withhydroxyl, to form an imine amide compound having the structural FormulaIV ##STR5##

(e) and thereafter hydrolyzing the imine amide product of step (d), ifdesired, in dilute aqueous acid to form an amide compound having thestructural Formula Va or Vb ##STR6## where R₄ ', R₅ ', Y', n', and W'are as previously defined; and

(f) converting the product of step (a), (b), (c), (d), or (e) to apharmaceutically acceptable salt, if desired.

DETAILED DESCRIPTION

In accordance with one subgeneric chemical compound aspect, the presentinvention provides esters of the formula ##STR7## where R₁ is methyl andR₃ is alkoxy of from one to four carbon atoms.

Particularly preferred is the compound6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester.

In another aspect, the present invention includes the compound6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid and itspharmaceutically acceptable salts.

In accordance with another subgeneric chemical compound aspect,compounds of the present invention include imine esters of structuralFormula IIIA or IIIB above where the group R₃ is alkoxy of from one tofour carbons and the imine group is defined as ═N--Y--NR₄ R₅ or═N(CH₂)_(n) --W. The groups R₄ and R₅ are independently hydrogen or arealkyl or hydroxyalkyl groups of from one to four carbon atoms, oralternatively, when R₃ is ═N(CH₂)_(n) '--W', n equals zero to four and Wis a pyrrolidinyl or piperidinyl ring optionally substituted with alkylof from one to four carbon atoms which may be further substituted withhydroxyl.

The linking group Y is cyclohexyl or is straight or branched alkylene offrom one to six carbon atoms, optionally substituted with hydroxyl. Bythe term "alkylene" as used throughout this specification and appendedclaims is meant a straight or branched hydrocarbon group of the generalformula C_(x) H_(2x) where x is an integer ranging from one to six. Whenx takes on a value of three to six, one or more of the hydrogen atoms ofthe linking group Y may be optionally substituted with hydroxyl.

The terms "alkyl" and "hydroxyalkyl" of from one to four carbon atoms asused throughout this specification and the appended claims are intendedto include straight chain and branched chain alkyl and hydroxyalkylgroups such as methyl, hydroxymethyl, ethyl, 2-hydroxyethyl, propyl, 2-and 3-hydroxypropyl, 1-methylethyl, 1-(hydroxymethyl)ethyl, butyl, 3-,and 4-hydroxybutyl, and the like.

Particularly preferred groups for R₄ and R₅ are hydrogen, methyl,hydroxymethyl, ethyl, 2-hydroxyethyl, propyl, 2- and 3-hydroxypropyl.

Examples of group Y include methylene, ethylene, propylene, butylene,pentylene, hexylene, 2-methylbutylene, 2-hydroxypropylene, 2-hydroxy-,and 2,3-dihydroxybutylene, cyclohexyl, and the like. Particularlypreferred groups for Y are methylene, ethylene, propylene,2-hydroxypropylene, and cyclohexyl.

Examples of compounds falling within the scope of this embodiment of theinvention include:

4,7,9-Trihydroxy-6-(iminomethyl)-1-phenazinecarboxylic acid, methylester.

4,7,9-Trihydroxy-6-(iminomethyl)-8-methyl-1-phenazinecarboxylic acid,methyl ester.

4,7,9-Trihydroxy-6-[(methylimino)methyl]-1-phenazinecarboxylic acid,methyl ester.

4,7,9-Trihydroxy-8-methyl-6-[(methylimino)methyl]-1-phenazinecarboxylicacid, methyl ester.

6-[[(2-Aminoethyl)]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxylicacid, methyl ester.

6-[[(2-Aminoethyl)]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid, methyl ester.

6-[[[2-(Dimethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxylicacid, methyl ester.

6-[[[2-(Dimethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid, methyl ester.

4,7,9-Trihydroxy-6-[[[2-(1-pyrrolidinyl)ethyl]imino]methyl]-1-phenazinecarboxylicacid, ethyl ester.

4,7,9-Trihydroxy-8-methyl-6-[[[2-(1-pyrrolidinyl)ethyl]imino]methyl]-1-phenazinecarboxylicacid, ethyl ester.

6-[[[4-(Dimethylamino)cyclohexyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxylicacid, methyl ester.

6-[[[4-(Dimethylamino)cyclohexyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid, methyl ester.

6-[[[(1-Ethyl-2-pyrrolidinyl)methyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxylicacid, methyl ester.

6-[[[(1-Ethyl-2-pyrrolidinyl)methyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid, methyl ester.

4,7,9-Trihydroxy-6-[[[2-(1-methyl-2-pyrrolidinyl)ethyl]imino]methyl]-1-phenazinecarboxylicacid, methyl ester.

4,7,9-Trihydroxy-8-methyl-6-[[[2-(1-methyl-2-pyrrolidinyl)ethyl]imino]methyl]-1-phenazinecarboxylicacid, methyl ester.

4,7,9-Trihydroxy-6-[[(1-methyl-4-piperidinyl)imino]methyl]-1-phenazinecarboxylicacid, methyl ester.

4,7,9-Trihydroxy-8-methyl-6-[[(1-methyl-4-piperidinyl)imino]methyl]-1-phenazinecarboxylicacid, methyl ester.

6-[[[5-(Dimethylamino)pentyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxylicacid, methyl ester.

6-[[[5-(Dimethylamino)pentyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid, methyl ester.

4,7,9-Trihydroxy-6-[[2-[(2-methylpropyl)amino]-ethyl]imino]methyl]-1-phenazinecarboxylicacid, methyl ester.

4,7,9-Trihydroxy-8-methyl-6-[[2-[(2-methylpropyl)amino]ethyl]imino]methyl]-1-phenazinecarboxylic acid, methyl ester.

6-[[[2-(Diethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxylicacid, methyl ester.

6-[[[2-(Diethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid, methyl ester.

4,7,9-Trihydroxy-6-[[[2-(methylamino)ethyl]-imino]methyl]-1-phenazinecarboxylicacid, methyl ester.

4,7,9-Trihydroxy-8-methyl-6-[[[2-(methylamino)ethyl]imino]methyl]-1-phenazinecarboxylicacid, methyl ester.

4,7,9-Trihydroxy-6-[[[2-[(2-hydroxyethyl)amino]ethyl]imino]methyl-1-phenazinecarboxylicacid, methyl ester.

4,7,9-Trihydroxy-6-[[[2-[(2-hydroxyethyl)amino]ethyl]imino]methyl-8-methyl-1-phenazinecarboxylicacid, methyl ester.

6-[[[2-(Ethylmethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxylicacid, methyl ester.

6-[[[2-(Ethylmethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid, methyl ester.

6-[[[2-[Bis(hydroxyethyl)amino]ethyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxylicacid, methyl ester.

6-[[[2-[Bis(hydroxyethyl)amino]ethyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid, methyl ester.

6-[[[3-(Dimethylamino)-2-hydroxypropyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxylicacid, methyl ester.

6-[[[3-(Dimethylamino)-2-hydroxypropyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid, methyl ester.

In accordance with another subgeneric chemical compound aspect,compounds of the present invention include imine acids of the structuralformula ##STR8## where Y, R₄, R₅, n, and W have the values definedabove.

Examples of compounds falling within the scope of this embodimentinclude:

4,7,9-Trihydroxy-6-(iminomethyl)-1-phenazinecarboxylic acid.

4,7,9-Trihydroxy-6-(iminomethyl)-8-methyl-1-phenazinecarboxylic acid.

6-[[(2-Aminoethyl)]imino]methyl]-4,7,9-trihydroxy -1-phenazinecarboxylicacid.

6-[[(2-Aminoethyl)]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid.

6-[[[4-(Dimethylamino)cyclohexyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxylicacid.

6-[[[4-(Dimethylamino)cyclohexyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid.

6-[[[(1-ethyl-2-pyrrolidinyl)methyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxylicacid.

6-[[[(1-ethyl-2-pyrrolidinyl)methyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid.

4,7,9-Trihydroxy-6-[[[2-(1-methyl-2-pyrrolidinyl)ethyl]imino]methyl]-1-phenazinecarboxylicacid.

4,7,9-Trihydroxy-8-methyl-6-[[[2-(1-methyl-2-pyrrolidinyl)ethyl]imino]methyl]-1-phenazinecarboxylic acid.

4,7,9-Trihydroxy-6-[[(1-methyl-4-piperidinyl)methyl]-1-phenazinecarboxylicacid.

4,7,9-Trihydroxy-8-methyl-6-[[(1-methyl-4-piperidinyl)imino]methyl]-1-phenazinecarboxylicacid.

6-[[[5-(Dimethylamino)pentyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxylicacid.

6-[[[5-(Dimethylamino)pentyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid.

4,7,9-Trihydroxy-6-[[[2-[(2-methylpropyl)amino]-ethyl]imino]methyl]-1-phenazinecarboxylicacid.

4,7,9-Trihydroxy-8-methyl-6-[[[2-[(2-methylpropyl)amino]ethyl]imino]methyl]-1-phenazinecarboxylicacid.

4,7,9-Trihydroxy-6-[[[2-(methylamino)ethyl]imino]methyl]-1-phenazinecarboxylicacid.

4,7,9-Trihydroxy-8-methyl-6-[[[2-(methylamino)ethyl]imino]methyl]-1-phenazinecarboxylicacid.

4,7,9-Trihydroxy-6-[[[2-[(2-hydroxyethyl)amino]ethyl]imino]methyl]-1-phenazinecarboxylicacid.

4,7,9-Trihydroxy-6-[[[2-[(2-hydroxyethyl)amino]ethyl]imino]methyl]-8-methyl-1-phenazinecarboxylicacid.

6-[[[2-(Ethylmethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxylicacid.

6-[[[2-(Ethylmethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid.

4,7,9-Trihydroxy-6-[[[2-(1-pyrrolidinyl)ethyl]imino]methyl]-1-phenazinecarboxylicacid.

4,7,9-Trihydroxy-8-methyl-6-[[[2-(1-pyrrolidinyl)ethyl]imino]methyl]-1-phenazinecarboxylicacid.

4,7,9-Trihydroxy-6-[[[2-(1-pyrrolidinyl)ethyl]imino]methyl]-1-phenazinecarboxylicacid.

4,7,9-Trihydroxy-6-[[[2-(1-pyrrolidinyl)ethyl]methyl]-8-methyl-1-phenazinecarboxylicacid.

6-[[[3-(Dimethylamino)-2-hydroxypropyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxylicacid.

6-[[[3-(Dimethylamino)-2-hydroxypropyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid.

In accordance with a further subgeneric chemical compound aspect,compounds of the present invention include imine amides of structuralFormula IV above where Y and Y' may be alike or different, R₄ and R₄ 'may be alike or different and R₅ and R₅ ' may be alike or different, nand n' may be alike or different and W and W' may be alike or different,and are as defined above.

Examples of compounds falling within this scope of the inventioninclude:

4,7,9-Trihydroxy-6-(iminomethyl)-1-phenazinecarboxamide.

4,7,9-Trihydroxy-6-(iminomethyl)-8-methyl-1-phenazinecarboxamide.

4,7,9-Trihydroxy-N-methyl-6-[(methylimino)methyl]-1-phenazinecarboxamide.

4,7,9-Trihydroxy-N,8-dimethyl-6-[(methylimino)methyl]-1-phenazinecarboxamide.

N-(2-Aminoethyl)-6-[[(2-aminoethyl)imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-(2-Aminoethyl)-6-[[(2-aminoethyl)imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

N-[2-(Dimethylamino)ethyl]-6-[[[2-(dimethylamino)ethyl]imino]methyl-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-[2-(Dimethylamino)ethyl]-6-[[[2-(dimethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

N-[2-(Dimethylamino)ethyl-6-[[[2-(diethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-[2-(Diethylamino)ethyl]-6-[[[2-(diethylamino)ethyl]imino]methyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

N-[3-(Dimethylamino)propyl]-6-[[[3-(dimethylamino)propyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-[3-(Dimethylamino)propyl]-6-[[[3-(dimethylamino)propyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-phenazinecarboxamide.

N-[2-[Bis(hydroxyethyl)amino]ethyl]-6-[[[2-[bis(hydroxyethyl)amino]ethyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-[2-[Bis(hydroxyethyl)amino]ethyl]-6-[[[2-[bis(hydroxyethyl)amino]ethyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

4,7,9-Trihydroxy-N-[2-(1-pyrrolidinyl)ethyl]-6-[[[2-(1-pyrrolidinyl)ethyl]imino]methyl]-1-phenazinecarboxamide.

4,7,9-Trihydroxy-8-methyl-N-[2-(1-pyrrolidinyl)ethyl]-6-[[[2-(1-pyrrolidinyl)ethyl]imino]methyl]-1-phenazinecarboxamide.

N-[2-(Dimethylamino)ethyl]-6-[[[3-(dimethylamino)-2-hydroxypropyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-[4-(Dimethylamino)cyclohexyl]-6-[[[4-(dimethylamino)cyclohexyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-[4-(Dimethylamino)cyclohexyl-[[[4-(dimethylamino)cyclohexyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

N-[(1-Ethyl-2-pyrrolidinyl)methyl]-6-[[[(1-ethyl-2-pyrrolidinyl)methyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-[(1-Ethyl-2-pyrrolidinyl)methyl]-6-[[[(1-ethyl-2-pyrrolidinyl)methyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

4,7,9-Trihydroxy-N-[2-(1-methyl-2-pyrrolidinyl)ethyl]-6-[[[2-(1-methyl-2-pyrrolidinyl)ethyl]imino]methyl]-1-phenazinecarboxamide.

4,7,9-Trihydroxy-8-methyl-N-[2-(1-methyl-2-pyrrolidinyl)ethyl]-6-[[[2-(1-methyl-2-pyrrolidinyl)ethyl]imino]methyl]-1-phenazinecarboxamide.

4,7,9-Trihydroxy-N-(1-methyl-4-piperidinyl)-6-[[(1-methyl-4-piperidinyl)imino]methyl]-1-phenazinecarboxamide.

4,7,9-Trihydroxy-8-methyl-N-(1-methyl-4-piperidinyl)-6-[[(1-methyl-4-piperidinyl)imino]methyl]-1-phenazinecarboxamide.

N-[5-(Dimethylamino)pentyl]-6-[[[5-(dimethylamino)pentyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-[5-(Dimethylamino)pentyl]-6-[[[5-(dimethylamino)pentyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

4,7,9-Trihydroxy-N-[2-[(2-methylpropyl)amino]ethyl]-6-[[[2-[(2-methylpropyl)amino]ethyl]imino]methyl]-1-phenazinecarboxamide.

4,7,9-Trihydroxy-8-methyl-N-[2-[(2-methylpropyl)amino]ethyl]-6-[[[2-[(2-methylpropyl)amino]ethyl]imino]methyl]-1-phenazinecarboxamide.

N-[2-(Diethylamino)ethyl]-6-[[[2-(Diethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-[2-(Diethylamino)ethyl]-6-[[[2-(Diethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

4,7,9-Trihydroxy-N-[2-(methylamino)ethyl]-6-[[[2-(methylamino)ethyl]imino]methyl]-1-phenazinecarboxamide.

4,7,9-Trihydroxy-8-methyl-N-[2-(methylamino)ethyl]-6-[[[2-(methylamino)ethyl]imino]methyl]-1-phenazinecarboxamide.

4,7,9-Trihydroxy-N-[2-[(2-hydroxyethyl)amino]ethyl]-6-[[[2-[(2-hydroxyethyl)amino]ethyl]imino]methyl]-1-phenazinecarboxamide.

4,7,9-Trihydroxy-N-[2-[(2-hydroxyethyl)amino]ethyl]-6-[[[2-[(2-hydroxyethyl)amino]ethyl]imino]methyl]-8-methyl-1-phenazinecarboxamide.

N-[2-(ethylmethylamino)ethyl]-6-[[[2-(ethylmethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-[2-(ethylmethylamino)ethyl]-6-[[[2-(ethylmethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

N-[2-(Dimethylamino)ethyl]-6-[[[3-(dimethylamino)-2-hydroxypropyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

In accordance with another subgeneric chemical compound aspect,compounds of the present invention include amides of Formula V where Y',R₄ ', and R₅ ' have the values given above.

Examples of compounds falling within this embodiment include:

6-Formyl-4,7,9-trihydroxy-1-phenazinecarboxamide.

6-Formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

6-Formyl-4,7,9-trihydroxy-N-methyl-1-phenazinecarboxamide.

6-Formyl-4,7,9-trihydroxy-N,8-dimethyl-1-phenazinecarboxamide.

N-(2-Aminoethyl)-6-formyl-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-(2-Aminoethyl)-6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

N-[2-(Dimethylamino)ethyl]-6-formyl-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-[2-(Dimethylamino)ethyl]-6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

N-[3-(Dimethylamino)propyl]-6-formyl-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-[3-(Dimethylamino)propyl]-6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

N-[2-[Bis(hydroxyethyl)amino]ethyl]-6-formyl-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-[2-[Bis(hydroxyethyl)amino]ethyl]-6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

6-Formyl-4,7,9-trihydroxy-N-[2-(1-pyrrolidinyl)ethyl]-1-phenazinecarboxamide.

6-Formyl-4,7,9-trihydroxy-8-methyl-N-[2-(1-pyrrolidinyl)ethyl]-1-phenazinecarboxamide.

N-[4-(Dimethylamino)cyclohexyl]-6-formyl-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-[4-(Dimethylamino)cyclohexyl]-6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

6-Formyl-4,7,9-trihydroxy-N-[2-(1-methyl-2-pyrrolidinyl)ethyl]-1-phenazinecarboxamide.

6-Formyl-4,7,9-trihydroxy-8-methyl-N-[2-(1-methyl-2-pyrrolidinyl)ethyl]-1-phenazinecarboxamide.

6-Formyl-4,7,9-trihydroxy-N-(1-methyl-4-piperidinyl)-1-phenazinecarboxamide.

6-Formyl-4,7,9-trihydroxy-8-methyl-N-(1-methyl-4-piperidinyl)-1-phenazinecarboxamide.

N-[5-(Dimethylamino)pentyl]-6-formyl-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-[5-(Dimethylamino)pentyl]-6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

6-Formyl-4,7,9-trihydroxy-N-[2-[(2-methylpropyl)amino]ethyl]-1-phenazinecarboxamide.

6-Formyl-4,7,9-trihydroxy-8-methyl-N-[2-[(2-methylpropyl)amino]ethyl]-1-phenazinecarboxamide.

N-[2-(Diethylamino)ethyl-6-formyl-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-[2-(Diethylamino)ethyl-6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

6-Formyl-4,7,9-trihydroxy-N-[2-(methylamino)ethyl]-1-phenazinecarboxamide.

6-Formyl-4,7,9-trihydroxy-8-methyl-N-[2-(methylamino)ethyl]-1-phenazinecarboxamide.

6-Formyl-4,7,9-trihydroxy-N-[2-[(2-hydroxyethyl)amino]ethyl]-1-phenazinecarboxamide.

6-Formyl-4,7,9-trihydroxy-N-[2-[(2-hydroxyethyl)amino]ethyl]-8-methyl-1-phenazinecarboxamide.

N-[2-(Ethylmethylamino)ethyl]-6-formyl-4,7,9-trihydroxy-1-phenazinecarboxamide.

N-[2-(Ethylmethylamino)ethyl]-6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.

Compounds in accordance with the present invention are prepared from thestarting materials 6-formyl-4,7,9-trihydroxy-1-phenazinecarboxylic acid,methyl ester or from6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester. The former compound,6-formyl-4,7,9-trihydroxy-1-phenazinecarboxylic acid, methyl ester, isknown variously in the chemical literature as lomofungin or lomondomycinand is produced in isolable quantities from fermentation broth mixturescontaining the microorganism Streptomyces lomondensis NRRL 3252 asdetailed in U.S. Pat. No. 3,359,165.

The latter compound,6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester, is produced in isolable quantities from fermentation brothmixtures containing the microorganism Streptomyces galanosa, designatedNRRL 15738 as described more fully below in Example 1.

Higher alkyl esters derived from the two starting methyl esters areobtained by conventional transesterification reactions using the methylesters as starting materials and followed, if necessary, by conventionalhydrolysis of any acetal or hemiacetal formed at the 6-formylfunctionality during the transesterification reaction.

The free acid forms of both compounds can be obtained by conventionalbasic hydrolysis of the methyl ester starting materials or the higheralkyl esters resulting from the transesterification in, for example,aqueous sodium hydroxide, followed by acidification.

Imine amide derivatives of Formula IV above are obtained, when R₁ ismethyl, by reaction of6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester with the desired amine, at a temperature of between about 25° C.and 100° C., preferably between about 30° C. and 70° C. Under theseconditions, the Schiff base imine forms first as a result of thereaction between the amine and the formyl group at position six of thephenazine ring system. The carbomethoxy functionality of the imine thusformed reacts further with the amine reagent to produce the imine amide.The product to be isolated is then purified by conventional chemicalmethods.

When R₁ is hydrogen, the preceding reaction sequence does not producethe imine amide in significant quantities, presumably because ofketo-enol tautomerism involving the hydrogen atom at position eight ofthe phenazine ring system and one of the adjacent phenolic groups. Toprepare the imine amides of Structure IV above where R₁ is hydrogen,6-formyl-4,7,9-trihydroxy-1-phenazinecarboxylic acid, formed byhydrolysis of the methyl ester starting material, is reacted with acoupling reagent such as 1,1'-carbonyldiimidazole or1,3-dicyclohexylcarbodiimide in an anhydrous polar organic solvent suchas dimethylformamide followed by the addition of the desired amine. Thereaction is carried out at temperatures between about -10° C. and 25° C.Under these conditions, if an excess of amine is used both the 6-formylfunctionality and the carboxylic acid functionality are derivatized byreaction with the amine to produce the imine amide.

The imine ester compounds of Formula IIIa or IIIb above, where R₃ isalkoxy, are prepared by reacting the appropriate6-formyl-4,7,9-trihydroxy-1-phenazine carboxylic acid ester or6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid ester withthe desired amine under mild conditions. For example, the esters may bereacted with the appropriate amine in a polar organic solvent such astetrahydrofuran or dimethylformamide at room temperature for a fewminutes, followed by isolation and purification of the product byconventional means.

Imine acids are prepared by reacting the6-formyl-4,7,9-trihydroxy-1-phenazinecarboxylic acid or6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid with thedesired amine under mild conditions, or alternatively, by basichydrolysis of the ester functionality of the corresponding imine esterswith, for example, dilute aqueous sodium hydroxide, followed byacidification.

The amides of Formula Va and Vb above are prepared by acidic hydrolysisof the imino functionality of the imine amides, prepared as describedabove with, for example, dilute aqueous hydrochloric acid followed byisolation and purification of the amide product by conventional means.

Compounds of the present invention, form salts with a number ofpharmaceutically acceptable inorganic bases and organic amines. Suitableinorganic bases include ammonium hydroxide and the hydroxides,carbonates, or bicarbonates of sodium, potassium, calcium, magnesium,iron, and zinc. Particularly preferred inorganic salts of compounds ofthe present invention are the sodium and potassium salts. Suitableorganic amines for preparing base addition salts of acidic compounds ofthe present invention form a class of amines well known to practitionersof the pharmaceutical formulation art.

The salts are prepared by contacting the compounds with an equivalentamount of the desired base in the conventional manner. The compounds maybe regenerated from the salts, if desired, by treating the salt with adilute aqueous solution of an acid such as hydrochloric.

Compounds of the present invention, when R₂ and/or R₃ contain a basicnitrogen atom, form salts with a number of pharmaceutically acceptableinorganic and organic acids which include hydrochloric, hydrobromic,hydriodic, sulfuric, nitric, phosphoric, acetic, benzoic, citric,maleic, malic, tartaric, succinic, gluconic, ascorbic, sulfamic, oxalic,pamoic, methanesulfonic, benzenesulfonic, and related acids and mixturesthereof.

The acid addition salts may be made by mixing the free base form of thecompound with the desired acid in a suitable solvent such as water oraqueous alcohol. The resulting solution may be evaporated or lyophilizedto recover the salt which may then be purified by conventional methods.

The free base form of the compound may be recovered from the acidaddition salt, if desired, by treating the salt with an aqueous solutionof a suitable base such as sodium hydroxide, sodium carbonate, and thelike.

The salts differ from the free base form of the compounds of thisinvention in such physical properties as melting point and solubility inpolar solvents, but are otherwise considered equivalent for the purposesof this invention.

The compounds of the present invention and their pharmaceuticallyacceptable salts can exist in unsolvated as well as solvated forms,including hydrated forms. In general, the solvated forms withpharmaceutically acceptable solvents such as water, ethanol, and thelike are considered equivalent to the unsolvated forms for the purposesof this invention.

The compounds of the present invention and their pharmaceuticallyacceptable salts are useful as antimicrobial agents. The antimicrobialactivity of the representative examples of compounds in accordance withthe present invention against five species of gram-negative bacteria,seven species of gram-positive bacteria, four species of yeast, and twospecies of fungi were determined using the microtiter dilutiontechnique. This method is described by T. B. Conrath, "Handbook ofMicrotiter Procedures," Dynatech Corp., Cambridge, Mass., USA (1972);and T. L. Gavan et al, "Microdilution Test Procedures," in Manual ofClinical Microbiology, E. H. Lennette, Ed., American Soc. forMicrobiol., Washington, D.C., U.S.A. (1980).

Each agent is suspended in a nonaqueous solvent for several minutes tosterilize the compound or, if the compound is completely soluble inwater, the aqueous solution is sterilized by passage through a 0.2-0.45micrometer membrane filter.

Each well of a sterile 96-well microdilution tray is filled underaseptic conditions with 0.1 ml of Mueller-Hinton broth (forantibacterial tests), or yeast extract-peptone-dextrose or bufferedsupplemented yeast nitrogen base (for tests with yeasts or fungi).

A 0.5-ml sample of the test compound solution is added to each of theeight wells in the first row of the tray. A microdilutor apparatus isused to simultaneously mix the contents of these wells and to transferaliquots to each succeeding row of cells to obtain a range of seriallydiluted solutions. The last row of wells is untreated and serves as acontrol.

Each well containing broth and test compound is inoculated with aboutten microliters of inoculum of the test microorganism. One well in thelast row of wells (which is free of test compound) is not inoculated andis used as a sterility control The trays are sealed and incubated at 37°C. for 16-24 hours (for bacteria) or at 28° C. for 36-48 hours (yeastsor fungi). During incubation, the inoculated medium is shaken at 100-140rpm to increase contact between the microorganism cells and testcompounds.

After the incubation period, each plate is evaluated by determining thelowest concentration of test compound required to inhibit the growth ofthe microorganism and recorded as a minimal inhibitory concentration(MIC). MIC values of <0.005 mg/ml to 0.333 mg/ml indicate antimicrobialactivity; values of 0.333-1.0 mg/ml are considered indicative ofmarginal activity; and MIC values of >1.0 mg/ml are consideredindicative of lack of activity against a particular microorganism

The results of these tests appear in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    Antimicrobial Activity                                                                      Minimal Inhibitory Concentration (mg/ml)                                      For Compound of Example:                                        Microorganisms                                                                              3     5     7    8    11   14   16   18                         __________________________________________________________________________    Escherichia coli (PD-04863)                                                                 >1.000                                                                              0.0123                                                                              0.111                                                                              1.000                                                                              0.111                                                                              0.111                                                                              0.037                                                                              0.037                      Salmonella typhimurium                                                                      1.000 0.0123                                                                              0.333                                                                              0.111                                                                              0.111                                                                              0.111                                                                              0.037                                                                              0.111                      Alcaligenes viscolactis                                                                     0.333 0.0041                                                                              0.0041                                                                             0.0041                                                                             0.0123                                                                             0.0123                                                                             0.0123                                                                             0.0123                     Branhamella catarrhalis                                                                     0.111 0.0014                                                                              0.0123                                                                             0.0041                                                                             0.0123                                                                             0.0123                                                                             0.0123                                                                             0.0123                     Pseudomonas aeruginosa                                                                      >1.000                                                                              0.111 <1.000                                                                             >1.000                                                                             1.000                                                                              1.000                                                                              0.333                                                                              >1.000                     Micrococcus luteus                                                                          0.111 0.0041                                                                              0.037                                                                              1.000                                                                              0.111                                                                              0.111                                                                              0.0123                                                                             0.0123                     Staphylococcus aureus                                                                       >1.000                                                                              0.0123                                                                              0.333                                                                              1.000                                                                              0.037                                                                              0.037                                                                              0.037                                                                              0.037                      Streptococcus pyogenes                                                                      0.111 0.0041                                                                              0.111                                                                              1.000                                                                              0.111                                                                              0.111                                                                              0.037                                                                              0.037                      Streptococcus pneumoniae                                                                    <0.00046                                                                            <0.00046                                                                            0.037                                                                              1.000                                                                              0.111                                                                              0.111                                                                              0.037                                                                              0.037                      Streptococcus faecalis                                                                      1.000 0.0123                                                                              1.000                                                                              >1.000                                                                             0.111                                                                              0.111                                                                              0.111                                                                              0.037                      Bacillus cereus                                                                             0.333 0.0014                                                                              0.111                                                                              0.037                                                                              0.037                                                                              0.037                                                                              0.0123                                                                             0.0123                     Bacillus megaterium                                                                         1.000 0.0041                                                                              0.333                                                                              1.000                                                                              0.111                                                                              0.111                                                                              0.037                                                                              0.037                      Saccharomyces cerevisiae                                                                    >1.000                                                                              0.111 1.000                                                                              >1.000                                                                             0.111                                                                              >1.000                                                                             0.111                                                                              0.111                      Schizosaccharomyces pombe                                                                   >1.000                                                                              0.037 0.111                                                                              >1.000                                                                             0.037                                                                              0.111                                                                              0.037                                                                              0.037                      Rhodotorula aurantiaca                                                                      >1.000                                                                              0.111 0.111                                                                              >1.000                                                                             0.0123                                                                             0.037                                                                              0.037                                                                              0.037                      Torulopsis albida                                                                           1.000 0.333 1.000                                                                              >1.000                                                                             0.111                                                                              0.333                                                                              0.037                                                                              0.037                      Mucor paraciticus                                                                           1.000 0.037 0.333                                                                              >1.000                                                                             0.037                                                                              0.333                                                                              0.111                                                                              0.037                      Rhizopus japonicus                                                                          >1.000                                                                              0.111 0.333                                                                              >1.000                                                                             0.037                                                                              0.333                                                                              0.111                                                                              0.037                      __________________________________________________________________________

The compounds of the present invention are also useful for inhibitingthe growth of neoplasma in mammals by virtue of their cytotoxic activityagainst such neoplastic growths.

The antineoplastic activities of representative compounds of the presentinvention against the L1210 murine leukemia cell line in vitro weredetermined using the methods detailed by R. I. Geran et al, "Protocolsfor Screening Chemical Agents and Natural Products Against Animal Tumorsand Other Biological Systems," 3rd Edition, Cancer Chemotherapy Reports,Part 3, Vol. 3, pages 1-87 (1972) which is incorporated herein byreference.

From duplicate tests, the cytotoxic activities were shown to be thosegiven in Table 2. The IC₅₀ values are the concentration of compoundrequired to produce 50% inhibition of growth of the cell line.

                  TABLE 2                                                         ______________________________________                                         Cytotoxic Activity Against L1210                                             Murine Leukemia In Vitro                                                      Compound of    IC.sub.50 (micro                                               Example        grams/ml)                                                      ______________________________________                                        3              0.17                                                           5              0.17                                                           7              0.63                                                           8              0.24                                                           11             3.47                                                           16             0.33                                                           18             0.24                                                           ______________________________________                                    

The in vivo antineoplastic activity of representative compounds of thepresent invention against the transplanted P388 leukemia cell line inmice was determined in accordance with the protocol given in Geran etal, cited above.

The mice were infected intraperitoneally on Day 0 and then administeredthe indicated doses of the test compounds on Days 1-9. The data arepresented in Table 3.

                                      TABLE 3                                     __________________________________________________________________________    Cytotoxic Activity Against P388                                               Murine Leukemia In Vivo                                                       % T/C* (Dosage; mg/kg/injection)                                              For Compound of Example Number                                                3     5     7    8     9    16    18                                          __________________________________________________________________________    Toxic (25)                                                                          137 (150)                                                                           166 (200)                                                                          205 (25)                                                                            219 (80)                                                                           213 (150)                                                                           132 (100)                                   149 (12.5)                                                                          113 (75)                                                                            152 (100)                                                                          146 (12.5)                                                                          170 (40)                                                                           166 (75)                                                                            123 (50)                                    130 (6.25)                                                                          104 (37.5)                                                                          140 (50)                                                                           129 (6.25)                                                                          144 (20)                                                                           147 (37.5)                                                                          106 (25)                                    119 (3.12)  126 (25)                                                                           113 (3.12)                                                                          143 (10)                                                                           136 (19)                                                                 139 (5)                                                __________________________________________________________________________     ##STR9##                                                                 

For use as antimicrobial or antineoplastic agents, the compounds of thepresent invention are formulated together with a compatible andpharmaceutically acceptable carrier in a form appropriate for thedesired route of administration. Examples of such forms include solidforms for oral administration such as tablets, pills, powders, andgranules, liquid forms for topical or oral administration such assolutions, emulsions, elixirs, and syrups, and forms suitable forparenteral administration such as sterile solutions, suspensions, oremulsions.

For preparing pharmaceutical compositions from the compounds describedby this invention, inert, pharmaceutically acceptable carriers can beeither solid or liquid. Solid form preparations include powders,tablets, dispersible granules, capsules, cachets, and suppositories. Asolid carrier can be one or more substances which may also act asdiluents, flavoring agents, solubilizers, lubricants, suspending agents,binders, or tablet disintegrating agents; it can also be anencapsulating material. In powders, the carrier is a finely dividedsolid which is in admixture with the finely divided solid of the activecompound. In the tablet the active compound is mixed with carrier havingthe necessary binding properties in suitable proportions and compactedin the shape and size desired. The powders and tablets preferablycontain from 5 or 10 to about 70 percent of the active ingredient.Suitable solid carriers are magnesium carbonate, magnesium stearate,talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoabutter, and the like. The term "preparation" is intended to include theformulation of the active compound with encapsulating material ascarrier providing a capsule in which the active component (with orwithout other carriers) is surrounded by carrier, which is thus inassociation with it. Similarly, cachets are included. Tablets, powders,cachets, and capsules can be used as solid dosage forms suitable fororal administration.

For preparing suppositories, a low melting wax such as a mixture offatty acid glycerides or cocoa butter is first melted, and the activeingredient is dispersed homogeneously therein as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions.As an example may be mentioned water or water-propylene glycol solutionsfor parenteral injection. Liquid preparations can also be formulated insolution in aqueous polyethylene glycol solution. Aqueous solutionssuitable for oral use can be prepared by dissolving the active componentin water and adding suitable colorants, flavors, stabilizing, andthickening agents as desired. Aqueous suspensions suitable for oral usecan be made by dispersing the finely divided active component in waterwith viscous material, i.e., natural or synthetic gums, resins,methylcellulose, sodium carboxymethylcellulose, and other well-knownsuspending agents.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for eitheroral or parenteral administration. Such liquid forms include solutions,suspensions, and emulsions. These particular solid form preparations aremost conveniently provided in unit dose form and as such are used toprovide a single liquid dosage unit. Alternately, sufficient solid maybe provided so that after conversion to liquid form, multiple individualliquid doses may be obtained by measuring predetermined volumes of theliquid form preparation as with a syringe, teaspoon, or other volumetriccontainer. When multiple liquid doses are so prepared, it is preferredto maintain the unused portion of said liquid doses at low temperature(i.e., under refrigeration) in order to retard possible decomposition.The solid form preparations intended to be converted to liquid form maycontain, in addition to the active material, flavorants, colorants,stabilizers, buffers, artificial and natural sweeteners, dispersants,thickeners, solubilizing agents, and the like. The liquid utilized forpreparing the liquid form preparation may be water, isotonic water,ethanol, glycerine, propylene glycol, and the like as well as mixturesthereof. Naturally, the liquid utilized will be chosen with regard tothe route of administration, for example, liquid preparations containinglarge amounts of ethanol are not suitable for parenteral use.

Preferably, the pharmaceutical preparation is in unit dosage form. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, for example, packeted tablets, capsules, and powders invials or ampoules. The unit dosage form can also be a capsule, cachet,or tablet itself or it can be the appropriate number of any of these inpackaged form.

The quantity of active compound in a unit dose of preparation may bevaried or adjusted from 0.1 mg to 500 mg preferably to 5 to 100 mgaccording to the particular application and the potency of the activeingredient. The compositions can, if desired, also contain othercompatible therapeutic agents.

In therapeutic use, the mammalian dosage range for a 70 kg subject isfrom 1 to 1500 mg/kg of body weight per day or preferably 2 to 750 mg/kgof body weight per day. The dosages, however, may be varied dependingupon the requirements of the patient, the severity of the conditionbeing treated, and the compound being employed. Determination of theproper dosage for a particular situation is within the skill of the art.Generally, treatment is initiated with smaller dosages which are lessthan the optimum dose of the compound. Thereafter the dosage isincreased by small increments until the optimum effect under thecircumstances is reached. For convenience, the total daily dosage may bedivided and administered in portions during the day if desired.

For use as antimicrobial agents, the compositions are administered sothat the concentration of the active component exceeds that required forthe minimal inhibition of the particular microorganism sought to becontrolled.

For use in inhibiting the growth of neoplasms in a mammal, thecompositions are administered parenterally in a daily dose which exceedsthe minimal concentration required for cytotoxicity of the particularneoplastic tissue sought to be controlled.

The following examples are provided to enable one skilled in the art topractice the present invention. These examples are not to be read aslimiting the scope of the invention as it is defined by the appendedclaims, but merely as illustrative thereof.

EXAMPLE 1 Fermentative production of6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester-50 liter scale

The actinomycete organism of the present invention which produces6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester was obtained from a soil sample collected in the Virgin Islands.The organism was isolated from the soil sample by standard platingtechniques using a suitable agar medium containing inorganic salts suchas sodium chloride, calcium carbonate, magnesium sulfate, and ferroussulfate, carbon substrates such as glycerol, and nitrogen sources suchas L-arginine. The organism was plated onto the agar medium andincubated at a favorable temperature, particularly 33° C., to allow fordevelopment of the organism

The microorganism has been designated: Streptomyces galanosa (NRRL15738) A comparison of the morphological characteristics and carbonutilization of the microorganism of this invention and those ofStreptomyces lomondensis (NRRL 3252) disclosed in U.S. Pat. No.3,359,165 appears in Tables 4 and 5, respectively.

                  TABLE 4                                                         ______________________________________                                                     Streptomyces  Streptomyces                                                    galanosa      lomondensis                                        Property     (NRRL 15738)  (NRRL 3252)                                        ______________________________________                                        Cell wall ornamen-                                                                         Rough to warty                                                                              Spiny                                              tation                                                                        Whole Cell analysis                                                                        L,L-Diaminopime-                                                                            L,L-Diaminopime-                                                lic acid, no  lic acid, no                                                    characteristic                                                                              characteristic                                                  sugar         sugar                                              Spore chain moro-                                                                          Long, tight   Mostly straight                                    phology      spirals, few  and open                                                        open and      spirals, few                                                    straight spirals                                                                            tight spirals                                      Gelatin lique-                                                                             Positive-brown                                                                              Positive-brown                                     faction                                                                       Milk coagulation                                                                           Negative      Negative                                           Milk peptonization                                                                         Negative-brown                                                                              Negative-light                                                  pigment       brown pigment                                      Reduction of Positive      Negative                                           nitrate                                                                       Melanine production                                                                        ISP1-Positive ISP1-Negative                                      or soluble   ISP6-Positive ISP6-Positive                                      pigments     ISP2-Light tan                                                                              ISP2-Russet                                                                   orange                                                          ISP3-Pearl pink                                                                             ISP3-Pearl                                                                    pink                                                            ISP4-Colorless                                                                              ISP4-Pastel                                                                   orange                                                          ISP5-Colorless                                                                              ISP5-Pastel                                                                   orange                                                          CIM23-Light tan                                                                             CIM23-Light                                                                   tan                                                Aerial mycelia                                                                             ISP2-Aqua grey                                                                              ISP2-Aqua grey                                                  ISP3-Slight white                                                                           ISP3-Slight                                                                   aqua grey                                                       ISP4-Aqua grey                                                                              ISP4-Aqua grey                                                  ISP5-Aqua grey                                                                              ISP5-Aqua grey                                                  CIM23-Aqua grey                                                                             CIM23-Aqua grey                                    Substratal mycelia                                                                         ISP2-Clove brown                                                                            ISP2-Brown                                                                    mahogany                                                        ISP3-Butterscotch                                                                           ISP3-Maple                                                      ISP4-Coffee   ISP4-Dark brown                                                 ISP5-Orange rust                                                                            ISP5-Wine                                                       CIM23-Clove   CIM23-Clove                                                     brown         brown                                              ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                                     Streptomyces                                                                              Streptomyces                                                      galanosa    lomondensis                                          Substrate    (NRRL 15738)                                                                              (NRRL 3252)                                          ______________________________________                                        L-Arabinose  +           +                                                    D-Fructose   +           +                                                    D-Galactose  +           +                                                    D-Glucose    +           +                                                    i-Inositol   +           +                                                    Inulin       +           +                                                    Maltose      +           +                                                    D-Mannitol   +           +                                                    Melobiose    +           +                                                    Raffinose    +           +                                                    Rhamnose     +           +                                                    Salicin      -           -                                                    Sucrose      +           +                                                    D-Xylose     +           +                                                    ______________________________________                                    

A culture of Streptomyces galanosa NRRL 15738, the microorganism of thepresent invention, has been deposited under the terms of the BudapestTreaty for the Deposit of Microorganisms with the Northern RegionalResearch Laboratory of the United States Department of Agriculture,Peoria, Ill. 61604, where it is being maintained in their permanentculture collection. The deposit has been designated NRRL 15738.

This microorganism is also maintained as a dormant culture in lyophiletubes, in cryogenic vials, and in soil sample tubes in theWarner-Lambert/Parke-Davis Culture Collection, 2800 Plymouth Rd., AnnArbor, Mich. 48105, where it bears the deposit designation WP-4611.

The compound, 6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid, methyl ester, which exhibits both antimicrobial and antitumoractivity is produced in isolable quantities by isolate NRRL 15738 duringaerobic fermentation under controlled conditions. The fermentationmedium consists of sources of carbon, nitrogen, minerals, and growthfactors. Examples of suitable carbon sources are glycerol and varioussimple sugars such as glucose, mannose, fructose, xylose, ribose, orother carbohydrate-containing substances such as dextrin, starch, cornmeal, and whey. The normal quantity of carbon source material in theculture medium generally varies between about 0.1 to 10 weight percent.

Suitable nitrogen sources for the culture medium include inorganic ormixed inorganic-organic nitrogenous compounds. Examples of suchmaterials are cottonseed meal, soybean meal, corn germ flour, corn steepliquor, distiller solubles, peanut meal, peptonized milk, and variousammonium salts.

The addition of minerals and growth factors to the fermentation broth isalso helpful in the fermentative production of6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester by isolate NRRL 15738. Examples of suitable minerals for theculture medium are potassium dihydrogen phosphate, sodium chloride,ferrous sulfate, calcium carbonate, cobalt chloride, and zinc sulfate.Growth factors are provided by such sources as various yeast and milkby-products.

The preferred method of producing6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester in accordance with the present invention is by submerged culturefermentation of a nutrient broth by isolate NRRL 15738. According tothis embodiment of the invention, the fermentation medium ingredientsare prepared in suspension and the resulting suspension is adjusted to apH value preferably between about pH 4 and pH 8. The culture medium isthen sterilized by autoclaving or steam heating, cooled to a suitabletemperature between 16° and 45° C., and then inoculated with themicroorganism.

Fermentation is then carried out with aeration and agitation untilisolable quantities of6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester are produced, usually within two to ten days under theseconditions.

In the submerged culture method, fermentation is carried out inshake-flasks or in stationary tank fermentors. In shake-flasks, aerationis effected by agitation of the flasks, causing efficient mixing of theinoculated medium with air. In stationary tank fermentors, agitation isprovided by impellers in the form of disk turbines, vaned discs, openturbines, or marine propellors. Aeration is provided by sparging air oroxygen into the fermentation mixture during agitation.

Isolate NRRL 15738 in its dormant state was transferred to an agar slanttube containing sterile CIM 23 culture medium and incubated for 7 to 14days at 28° C.

                  TABLE 6                                                         ______________________________________                                         Composition of CIM 23 Culture Medium                                         ______________________________________                                        Amidex Corn Starch       20     g                                             N-Z Amine, Type A        2      g                                             Beef Extract (Difco)     1      g                                             Yeast Extract (Difco)    1      g                                             Cobaltous chloride hexahydrate                                                                         20     mg                                            Agar                     20     g                                             Distilled water          1000   ml                                            ______________________________________                                    

A portion of the resulting microbial growth was used to inoculate an18×150 mm tube containing 5 ml of SD-05 culture medium. The seed tubeculture was shaken at 33° C. on a rotary gyratory shaker at 170 rpm forthree to four days.

                  TABLE 7                                                         ______________________________________                                         Composition of SD-05 Culture Medium                                          ______________________________________                                        Yeast Extract (Amberex 1003)                                                                           5      g                                             Glucose monohydrate (cerelose)                                                                         1      g                                             Dextrin (Amidex B 411, Corn                                                                            24     g                                             Products Co.)                                                                 Casein Digest (N-Z Case, Sheffield)                                                                    5      mg                                            Spray-dried Meat Solubles (Daylin                                                                      3      g                                             Labs)                                                                         Calcium carbonate        2      g                                             Distilled Water          1000   ml                                            ______________________________________                                    

A 1-ml portion of the microbial growth from the seed tube wastransferred to a 38×200 mm shake-tube containing 25 ml of PM-12screening medium. The PM-12 screening medium consisted of a watersuspension of 1% dextrin and 1% by weight of a feed grade mixturecomposed of soybean meal, ground yellow corn, ground wheat, corn glutenmeal, wheat middlings, dried milk products, animal fat preserved withBHA, ground beet pulp, calcium carbonate, sucrose, dehydrated alfalfameal, dicalcium phosphate, brewer's dried yeast, salt, vitamin B₁₂supplement, riboflavin supplement, calcium pantothenate, niacin, cholinechloride, menadione sodium bisulfite (source of vitamin K activity),folic acid, pyridoxine hydorchloride, thiamin, ascorbic acid, vitamin Asupplement, vitamin D activated animal sterol (source of vitamin D₃),vitamin E supplement, iron carbonate, iron sulfate, calcium iodate,manganous oxide, copper oxide, cobalt carbonate, and zinc oxide.

The inoculated shake-tube was incubated for three days at 33° C. on agyratory shaker at 170 rpm (5 cm throw). The production of6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester was monitored by screening the fermentation broth againstEscherichia coli (PD-08000).

E. coli (PD-08000) was seeded onto agar plates and 6.35 mm diameterpaper discs impregnated with the fermentation beer were laid on theinoculated medium and incubated at 37° C. overnight. The diameters ofthe zones of inhibition around the paper discs indicated the relativeconcentration of6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester in the fermentation broth.

Isolate NRRL 15738 was used to inoculate a 2-liter baffled Erlenmeyerflask containing 600 ml of SD-05 culture medium. The flask contents wereincubated at 33° C. for 65-75 hours. The resulting microbial growth wasused to inoculate 16 liters of SD-05 culture medium contained in a30-liter stirred-jar fermentor. The contents of the stirred jar wereincubated at 33° C. for 24 hours with stirring and air sparging at arate of one volume/volume/minute. The resulting microbial growth wasused to inoculate four production scale stirred jar fermentors.

Four 30-liter stirred-jar fermentors, each containing about 16 liters ofPM-12 culture medium were sterilized by autoclaving for 90 minutes at121° C. The jar contents were then cooled to 33° C. and then inoculatedwith 800 ml of inoculum from the previously described batch. The fourjars were subsequently incubated at 33° C. for three days while beingstirred and sparged with air at a rate of one volume/volume/minute. Theproduction of 6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid, methyl ester was monitored throughout this fermentation by assayagainst E. coli (PD-08000) as described above. The data for these assaysappear in Table 8.

                  TABLE 8                                                         ______________________________________                                        Microbial Assay of Fermentative Production of                                 6-Formyl-4,7,9-trihydroxy-8-methyl-1-                                         phenazinecarboxylic acid, Methyl Ester                                                                    Inhibition Zone                                                    Sedimentation                                                                            Diameter (mm) for                                                  Value      12.7 mm Disk vs.                                  Time             (Percent   Escherichia coli                                  (hours) pH       Growth)    (PD-08000)                                        ______________________________________                                        Jar 1                                                                          0      6.6      --          0                                                27      6.3      6.0        24                                                50      6.4      4.7        23                                                71      7.2      4.7        23                                                Jar 2                                                                          0      6.6      --          0                                                27       6.35    6.7        23                                                50      6.6      4.7        22                                                71      7.2      5.3        25                                                Jar 3                                                                          0      6.6      --          0                                                27      6.2      5.3        23                                                50      6.4      4.7        22                                                71      7.0      4.0        23                                                Jar 4                                                                          0      6.6      --          0                                                27      6.2      6.0        24                                                50       6.45    4.7        24                                                71      7.0      4.7        24                                                ______________________________________                                    

EXAMPLE 2 Fermentative production of6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester--5000 liter scale

A cryogenically preserved sample containing approximately 1 ml ofStreptomyces galanosa NRRL 15738 culture was used to inoculate 600 ml ofSD-05 seed medium contained in a two-liter baffled shake flask. Theinoculated flask contents were incubated for 72 hours at 33° C. whilebeing shaken on a gyrating shaker at 130 rpm.

After 72 hours, the contents of the seed flask were transferredaseptically to a 30-liter seed medium. The inoculated fermentor contentswere incubated at 33° C. for 28 hours while being stirred at 300 rpm andsparged with air at a rate of 1 vol/vol/min.

The microbial growth from the above stainless steel fermentor was usedto inoculate 75 gallons (284 liters) of SD-05 seed medium contained in a200-gallon (757-liter) stainless steel fermentor. The medium wassterilized by steam heating at 121° C. for 40 minutes after which thefermentor and contents were cooled to 33° C. and inoculated with about15 liters of the microoganism-containing broth. The resulting mixturewas incubated at 33° C. for 23 hours while being stirred at 155 rpm andsparged with air at a rate of about 0.75 vol/vol/min.

After 23 hours the microbial growth from the above 200-gallon fermentorwas used to inoculate 1300 gallons (4921 liters) of PM-13 nutrientmedium contained in a 2000-gallon (7571-liter) stainless steelfermentor.

                  TABLE 9                                                         ______________________________________                                         Composition of PM-13 Nutrient Medium                                         ______________________________________                                        Ammonium Sulfate (Allied Chemical)                                                                   0.1%                                                   Calcium Carbonate (Pfizer)                                                                           0.4%                                                   Pharmamedia (Traders Protein)                                                                        2.0%                                                   Cerelose (Atlas Sugar) 2.0%                                                   Maltrin (Grain Processing)                                                                           2.0%                                                   No pH adjustment                                                              ______________________________________                                    

The medium was sterilized prior to inoculation by heating with steam for40 minutes at 121° C. After sterilization, the fermentor and contentswere cooled to 33° C., inoculated, and incubated for 27 hours at 33° C.with stirring at 125 rpm and air sparging at a rate of 0.75 vol/vol/min.

As in Example 1, the progress of the fermentation was followed byperiodically measuring pH, sedimentation vlues, and inhibition of thegrowth of E. coli (PD-08000). Additionally, the ability of thefermentation broth, at dilutions of 1:50 and 1:250, to inhibit the invitro growth of the L1210 murine leukemia cells was measured at severalpoints during the course of the fermentation. These data appear in Table10.

                  TABLE 10                                                        ______________________________________                                                    Sedimenta- Inhibition Zone                                                                           % Growth                                               tion Value Diameter (in mm,                                                                          Inhibition of                              Time        (Percent   12.7 mm disks) vs.                                                                        L1210 cells at                             (hrs) pH    Growth)    E. Coli (PD 08000)                                                                        1:50  1:250                                ______________________________________                                         0    7.1    7.4       --          --    --                                   23    5.7   20.0       25          100%  71%                                  24    5.6   18.7       26          100%  76%                                  26    5.5   19.3       23          100%  64%                                  27    5.4   16.7       24          100%  65%                                  ______________________________________                                    

EXAMPLE 3 Isolation of6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester

The unfiltered beer (4763 liters) from Example 2 above was adjusted topH 3.0 with 50% sulfuric acid. Celite 545 (660 kg) was added and themixture was stirred for one hour with 3030 liters of ethyl acetate.Additional sulfuric acid was added to maintain the pH at 3.0. Themixture was then filtered and the filter cake was washed twice with380-liter portions of ethyl acetate. The filtrate and washes werecombined and the upper ethyl acetate layer was concentrated in vacuo to337 liters. This concentrate was stored for 65 hours at -20° C. Theprecipitate that formed was filtered off, washed with three liters ofethyl acetate, four liters of methanol, and finally with 23.5 liters ofchloroform to leave a solid that was dried in vacuo. This partiallycrystalline product (155 g) was recrystallized from 18 liters ofchloroform:methanol (9:1) to afford 63 g of crystalline6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester. Concentration and cooling of the mother liquor afforded anadditional 23 g of crystalline product.

The product was analyzed by a high pressure liquid chromatographic(HPLC) method utilizing a 4.1 (ID)×250 mm PRP-1 column (Hamilton Co.,Reno, Nev. 89510) and a mobile phase consisting of a linear gradientfrom 0.025 M pH 9.5 borate buffer: acetonitrile:methanol (90:5:5) attime zero to 0.025 M pH 9.5 borate buffer:acetonitrile:methanol(70:25:5) over a course of seven minutes at a flow rate of 2.0 ml/min.The retention time of the product in this system is 3.5 minutes.

EXAMPLE 4 Preparation of6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester, potassium salt (phenolate salt)

6-Formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester (68.2 mg, 0.208 mmol) was suspended in 5 ml of water and asolution of 0.416 mmol of potassium hydroxide in 0.5 ml of water wasadded. The resulting red solution was lyophilized to produce 74.4 mg of6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester potassium salt as a rust-colored solid.

EXAMPLE 5 Preparation of6-[[[2-(dimethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid, methyl ester

6-Formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester (500 mg, 1.52 mmol) was suspended in 50 ml of tetrahydrofuran and2-(dimethylamino)ethylamine (160 mg, 1.83 mmol) was added dropwise withstirring to produce a deep purple solution. After about 20 minutes atroom temperature, an orange precipitate formed and the reaction mixturegradually developed an orange color.

The crude orange product was separated by filtration, washed withtetrahydrofuran, and dried under vacuum to yield 480 mg (80.3%) of6-[[[2-(dimethylamino)ethyl]imino]methyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid, methyl ester. The retention time for the product, employing theHPLC system and method described in Example 3 above was 5.2 minutes.

EXAMPLE 6 Preparation of6-[[[2-(dimethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid, methyl ester, methanesulfonate salt

6-[[[2-(Dimethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylicacid, methyl ester (468 mg, 1.177 mmol) was stirred at room temperaturewith methanesulfonic acid (125 mg, 1.3 mmol) in 20 ml of water untildissolution was complete.

The solution was diluted with water and lyophilized to yield the crudemethanesulfonate salt which was washed with isopropyl alcohol, andfiltered. The solid residue was dissolved in water and lyophilized toyield 423 mg of the purified salt.

EXAMPLE 7 Preparation ofN-[2-(dimethylamino)ethyl]-6-[[[2-(dimethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide

6-Formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester (1.0 g, 3.05 mmol) was dissolved in 50 ml of anhydrousN,N-dimethylethylenediamine (Aldrich Chemical Co., Milwaukee, Wis., USA)and stirred at 55° C. for 145 minutes under a nitrogen atmosphere. Thecourse of the reaction was followed by high pressure liquidchromatographic (HPLC) methods as described in Example 3.

After the reaction was complete, the excess N,N-dimethylethylenediaminewas evaporated under vacuum and the residue was dissolved in 150 ml ofwater. The pH of this solution was adjusted to 4.5 with acetic acid andthe resulting solution chromatographed over 300 ml of Diaion HP-20 resin(Mitsubishi Chem. Ind. Ltd., 5-2, Marunochi 2 Chiyodaku, Tokyo, Japan)which had been previously equilibrated with 0.075 M ammonium acetatebuffer (pH 4.5).

The column was washed with two 600-ml portions of ammonium acetatebuffer and then eluted with 200 ml portions of 5%, 10%, and 15%acetonitrile in buffer. The eluates were assayed by HPLC, and thosecontaining the product were combined and the acetonitrile removed byevaporation under vacuum. The remaining solution was lyophilized. Thelyophilate was triturated twice with 100-ml portions of chloroform. Thechloroform solution was filtered and extracted twice with 100-mlportions of water. The aqueous extracts were combined, the excesschloroform removed by evaporation, and the aqueous solution lyophilizedto yield 986 mg (2.31 mmol, 75.7%) ofN-[2-(dimethylamino)ethyl]-6-[[[2(dimethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide as a dark red,hygroscopic powder. The retention time of this product employing theHPLC system and method described in Example 3 above, was 5.6 minutes.

EXAMPLE 8 Preparation of N-[2-(dimethylamino)ethyl]-6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide

N-[2-(dimethylamino)ethyl]-6-[[[2-(dimethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide (252 mg, 0.59 mmol) wasdissolved in 50 ml of 0.3 M aqueous hydrochloric acid. The mixture wasstirred for three hours at 55° C. and the course of the reaction wasfollowed by the previously described HPLC methods.

When the reaction was complete, the mixture was diluted with 50 ml of10% sodium chloride solution and 50 ml of 1.0 M sodium phosphate buffer(pH 7.0) and extracted twice with 50-ml portions of 1-butanol.

The butanol extracts were combined and washed with 20 ml of water. Theextract was then concentrated under vacuum to remove excess water, andcooled to -20° C. for eighteen hours. Deep red crystals of the productformed, which were separated by filtration to yield 160 mg (0.42 mmol,71%) of N-[2-(dimethylamino)ethyl]-6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide, mp>250° C. The retentiontime, using the HPLC system and method described in Example 3, above,was 4.6 minutes.

EXAMPLE 9 Preparation of N-[2-(dimethylamino)ethyl]-6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide, methanesulfonate salt

N-[2-(Dimethylamino)ethyl]-6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide(5.75 g, 0.015 mole) was dissolved in 103 ml of 0.15 M methanesulfonicacid (Thiokol/Ventron) to give a clear red solution which wasfreeze-dried. The resulting orange-red solid was transferred to asintered glass funnel and washed with three volumes of isopropyl alcohol(100, 75, and 100 ml). The solid residue was then redissolved in 150 mlof water, filtered, and freeze-dried to give 6.73 g (94%) ofmethanesulfonate salt as an orange solid.

EXAMPLE 10 Conversion of N-[2-(dimethylamino)ethyl]-6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide methanesulfonate salt toalternative salt forms

The methane sulfonate salt prepared above in Example 9 is dissolved inwater and the pH adjusted to 5.5 with aqueous NaOH. After cooling at 5°C. overnight the free base form crystallizes from the solution and iscollected by filtration, washed with acetonitrile, and dried in vacuo.The product thus formed is then treated with an aqueous solutioncontaining one equivalent of the desired acid such as hydrochloric,gluconic, etc., and lyophilized to produce the new salt form.

EXAMPLE 11 Preparation of6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid

6-Formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester (500 mg, 1.52 mmol) was suspended in 20 ml of 1 M aqueous sodiumhydroxide solution and stirred at 60° C. for 1.2 hours.

The reaction mixture was acidified by dropwise addition of concentratedhydrochloric acid and the precipitate which formed was collected byfiltration. The residue was partially dissolved in ethylacetate:methanol and the mixture filtered. Upon standing at roomtemperature, crystals of 6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid formed which werecollected by filtration.

The residue which had not dissolved in ethyl acetate:methanol wasdissolved in boiling chloroform: methanol and the solution filtered.Upon refrigeration, additional crystals of6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid formedfrom the chloroform:methanol solution and were collected by filtration.

The two crops of crystals were combined to yield 150 mg of the acid. Theretention time, employing the HPLC system and method of Example 3 above,was 1.0 minute.

EXAMPLE 12 Preparation of6-formyl-4,7,9-trihydroxy-1-phenazinecarboxylic acid

6-Formyl-4,7,9-trihydroxy-1-phenazinecarboxylic acid, methyl ester (400mg, 1.27 mmol), prepared as described in U.S. Pat. No. 3,359,165 wasdissolved in 40 ml of 1 M aqueous sodium hydroxide solution and stirredfor 145 minutes at 25° C., with the reaction being monitored by highpressure liquid chromatographic techniques as described above in Example3. When the hydrolysis was complete, the solution was adjusted to pH 2.5by the addition of 1 M HCl whereupon a red precipitate of crude6-formyl-4,7,9trihydroxy-1-phenazinecarboxylic acid formed. After 15minutes, the precipitate was collected by filtration, washed with water,and dried. The crude acid was recrystallized from 200 ml of 1:1chloroform: methanol to yield 282 mg (0.94 mmol, 74%) of6-formyl-4,7,9-trihydroxy-1-phenazinecarboxylic acid. The retentiontime, employing the HPLC system and method described above in Example 3,was 1.3 minutes.

EXAMPLE 13 Preparation ofN-[2-(dimethylamino)ethyl]-6-[[[2-(dimethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxamide

6-Formyl-4,7,9-trihydroxy-1-phenazinecarboxylic acid (520 mg, 1.73mmoles) was suspended in 15 ml of dry dimethylformamide (DMF) and1,1'-carbonyldiimidazole (700 mg, 4.32 mmoles) dissolved in 4 ml of DMFwas added. The reaction mixture was stirred for 20 minutes at 25° C.until a clear solution had formed. The solution was then cooled in anice bath and N,N-dimethylethylenediamine (0.6 ml) added. After stirringfor 15 minutes at 25° C. the excess reagents were removed under vacuumto yieldN-[2-(dimethylamino)ethyl]-6-[[[2-(dimethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxamide. The retention time for thisproduct in the HPLC system described above in Example 3 is 5.0 minutes.

EXAMPLE 14 Preparation ofN-[2-(dimethylamino)ethyl]-6-formyl-4,7,9-trihydroxy-1-phenazinecarboxamid

The imine amide prepared above in Example 13 was dissolved in 425 ml ofwater and the pH adjusted to 6.5 with hydrochloric acid. After heatingto 45° C., 45 ml of concentrated hydrochloric acid was added slowly andthe solution was stirred at 45° C. for 80 minutes, cooled, and extractedthree times with ethyl acetate. The lower aqueous layer was concentratedin vacuo to remove residual organic solvent and then chromatographedover 100 ml of Diaion HP-20 resin. The column was washed with 500 ml of0.01 M HCl then eluted with acetonitrile:0.01 M HCl (1:1). The eluate(50 ml) was diluted with 50 ml of acetonitrile:water (1:1) and the pHadjusted to 5.5 with 1 M aqueous NaOH and cooled to 5° C. After 18 hoursthe red precipitate which had formed was separated by filtration, washedwith water and acetonitrile, and dried in vacuo to yield 530 mg ofN-[2-(dimethylamino)ethyl]-6-formyl-4,7,9-trihydroxy-1-phenazinecarboxamide,which could be further purified by recrystallization from hotchloroform-methanol (1:1). This product has a retention time in the HPLCsystem described above in Example 3, of 2.9 minutes.

EXAMPLE 15 Preparation ofN-[2-(diethylamino)ethyl]-6-[[[2-(diethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide

6-Formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester (1.02 g, 3.02 mmol) was stirred at 65° C. for two hours with 50 mlof 2-(diethylamino)ethyl amine. When the reaction was complete, asdetermined by HPLC analysis in the previously described system, theexcess amine was removed under vacuum to yieldN-[2-(diethylamino)ethyl]-6-[[[2-(diethylamino)ethyl]imino]methyl]-4,7,9-trihydroxy-1-phenazinecarboxamide. The retention time for thisproduct using the system described above in Example 3 is 6.0 minutes.

EXAMPLE 16 Preparation of N-[2-(diethylamino)ethyl]-6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide

The imine amide product from Example 15 was dissolved in 50 ml of waterand heated to 55° C. with stirring. Concentrated hydrochloric acid (6ml) was added dropwise and the temperature of the mixture was slowlyincreased to 72° C. Hydrolysis of the imine functionality wasessentially complete after two hours as indicated by HPLC analysis. Thereaction mixture was heated for an additional hour, and then cooled inice to produce the product as a yellow precipitate which was collectedby filtration.

The solid residue was dissolved in 0.01 N HCl (5 ml) and chromatographedover HP-20 (100 ml, previously equilibrated with 0.01 N HCl). The columnwas washed with 0.01 N HCl (200 ml) and eluted with water:acetonitrile(9:1, 100 ml) and water: acetonitrile (1:1, 100 ml). The eluatescontaining product were combined, concentrated in vacuo, thenlyophilized to yield 925 mg ofN-[2-(diethylamino)ethyl]-6-formyl-4,7,9-trihydroxy-1-phenazinecarboxamide.The retention time for this product in the HPLC system described abovein Example 3 is 4.5 minutes.

EXAMPLE 17 Preparation ofN-[3-(dimethylamino)propyl]-6-[[[3-(dimethylamino)propyl]imino]methyl]-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide

6-Formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester (1.107 g, 3.27 mmol), was stirred with 50 g of3-(dimethylamino)propyl amine (Aldrich Chemical Company, Milwaukee,Wis., USA) at 60° C. for 1.5 hours. The reaction mixture was then cooledin an ice bath for 20 minutes and the crude imine amide productprecipitated from solution. The precipitated solid was collected byfiltration and washed with 50 ml of acetonitrile. The retention time forthis product, employing the HPLC system and method described in Example3 above, was 4.6 minutes.

EXAMPLE 18 Preparation of N-[3-(dimethylamino)propyl]-6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide

An aqueous solution (50 ml) of the imine amide from Example 17 wasstirred under a nitrogen atmosphere while 2 ml of concentratedhydrochloric acid was added in a dropwise manner. During the course ofeight hours an additional 8 ml of concentrated hydrochloric acid (10 mltotal) was added. The reaction mixture was then cooled in an ice bathand the solution saturated with sodium chloride to induce precipitation.The precipitated amide was collected by filtration and washedconsecutively with acetonitrile and water. The residual orange solid wasdissolved in water and chromatographed over HP-20 resin (50 ml,previously equilibrated with 0.01 N HCl). The column was washed with0.01 N HCl, then eluted with 0.01 N HCl:acetonitrile (1:1). The eluatewas adjusted to pH 5.5 with aqueous NaOH, concentrated in vacuo toremove the acetonitrile, and then lyophilized to give 530 mg ofN-[3-(dimethylamino)propyl]-6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxamide.The retention time of this product, using the HPLC system describedabove in Example 3, is 3.6 minutes.

We claim:
 1. A purified culture of Streptomyces galanosa NRRL 15738which is capable of producing6-formyl-4,7,9-trihydroxy-8-methyl-1-phenazinecarboxylic acid, methylester in isolable quantities under conditions of aerobic fermentation ofa broth containing assimilable sources of carbon and nitrogen.